Genomic analysis using DNA sequence of random clones coupled with hybridization to electrophoretically separated DNA fragments has recently provided an unusual insight into the physiology, metabolism and evolutionary relationships of Mycoplasma genitalium (this laboratory). Of added significance is the fact that this highly fastidious organism possesses the smallest genome size of any free living species. With the knowledge that this methodology provides a valuable aid to assess phylogeny and taxonomy coupled with a powerful probe for analysis of the minimal essential genes required for independent self-replicating existence, we intend to expand this study to include classical M. fermentans (incognitus) plus other mycoplasmas, with special reference to those species having clinical significance to man. This approach will provide important clues to the metabolic pathways these organisms share, the nutrients a parasitic mode provides (missing biosynthetic pathways), and an aid for evaluation of the importance of various conserved genes and pathogenicity determinants. It will also reveal the chromosomal distribution of genes on the respective genomes and provide a new basis from which to assess the relatedness of mycoplasmas to each other and to higher gram positive prokaryotes. All of these studies can be accomplished with a few micrograms of DNA from each species, eliminating the necessity for repeated laboratory cultivation of these highly fastidious pathogens.